Configurational stereoisomer of difethialone, composition and rodenticidal bait comprising same, and process for controlling target rodent pests

ABSTRACT

Disclosed is a dextrorotatory enantiomer of the configurational stereoisomer of difethialone, named homo-stereoisomer, the formula of which is 3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene, in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group of the homo-stereoisomer have the same absolute configuration.

FIELD OF THE INVENTION

The invention relates to a configurational stereoisomer of difethialoneas an isolated compound, to a composition and a rodenticidal baitcomprising such a configurational stereoisomer and to a process forcontrolling target rodent pests. The invention thus relates to thetechnical field of controlling populations of target rodent pests.

BACKGROUND OF THE INVENTION

It is known practice to use poisons in the form of rodenticidal baitsagainst target rodent pests. It is known from EP 2 090 164 thatdifethialone is a second-generation anticoagulant acting in a singledose manner. US 2005/181003 describes a rodenticidal bait in gel formcomprising difethialone in a mass proportion of 25 ppm.

Such a bait is liable to be consumed by animals other than target rodentpests when it is made available to target rodent pests. It may beconsumed directly (primary consumption) by domestic animals or pets. Itmay also be consumed accidentally by humans. Such consumption may resultin poisoning, which may be lethal, of these domestic animals, pets orhumans.

In addition, a fraction of the difethialone of these rodenticidal baitsmay be ingested (secondary consumption) by animals—especially bybirds—which prey on weakened rodent pests that have consumed such arodenticidal bait, or by animals which carrion-feed on rodent pests thathave died from having consumed such a rodenticidal bait. This secondaryconsumption is liable in the long term to result in the death of thesepredatory or carrion-feeding animals, which may be animals—especiallybirds—belonging to protected species.

SUMMARY OF THE INVENTION

The invention is thus directed towards overcoming these drawbacks byproposing a configurational stereoisomer of difethialone, a compositionand a rodenticidal bait comprising such a configurational stereoisomerand a process for controlling harmful rodent pests, which are not onlyeffective for controlling the populations of target rodent pests but canalso limit the risks of poisoning of non-target animals—especiallydomestic or reared animals, pets or humans—which accidentally consumesuch a rodenticidal bait.

The invention is thus directed towards overcoming these drawbacks byproposing a configurational stereoisomer of difethialone, a compositionand a rodenticidal bait comprising such a configurational stereoisomerand a process for controlling target rodent pests, which are not onlyeffective for controlling the populations of target rodent pests but canalso limit the risks of secondary poisoning of wild animals—for examplefoxes or birds—which prey on weakened target rodent pests that haveconsumed the rodenticidal bait or of wild animals which carrion-feed ontarget rodent pests that died from being poisoned.

The invention is also directed towards proposing a configurationalstereoisomer of difethialone, a composition and a rodenticidal baitcomprising such a configurational stereoisomer and a process forcontrolling target rodent pests, the use of which is in accordance withthe rules of good practice, especially with respect to the protection ofbirds, and in particular birds of prey.

The invention is also directed towards proposing a configurationalstereoisomer of difethialone, a composition and a rodenticidal baitcomprising such a configurational stereoisomer and a process forcontrolling target rodent pests, which do not require, in order tocontrol a population of target rodent pests, the use of a rodenticidalagent at high dose and which are friendly towards the environment andthe health of humans and non-target animals—especially birds.

The invention is also directed towards proposing a configurationalstereoisomer of difethialone, a composition and a rodenticidal baitcomprising such a configurational stereoisomer and a process forcontrolling target rodent pests, which are able to be used forcontrolling target rodent pests that are resistant to known baits forcontrolling target rodent pests.

The invention is thus directed towards proposing an alternative to knownrodenticidal baits.

To do this, the invention relates to a dextrorotatory enantiomer of aconfigurational stereoisomer of difethialone, named homo-stereoisomer,the formula of which is3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group ofsaid homo-stereoisomer have the same absolute configuration.

Throughout the text:

-   -   the term “difethialone” denotes the compound        3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene        or        3-[3-[4-(4-bromophenyl)phenyl]-1-tetralinyl]-2-hydroxy-4-thiochromenone        or 3-[3-(4′-bromo        [1,1′-biphenyl]-4-yl)-1,2,3,4-tetrahydro-1-naphthalenyl]-4-hydroxy-2H-1-benzothiopyran-2-one        of formula (I) below:

in which the numbers of carbons 1 and 3 of the1,2,3,4-tetrahydronaphthalene group are indicated;

-   -   the term “stereoisomers” denotes isomers of the same        semi-structural formula, but in which the relative position of        the atoms differs in space. The term “configurational        stereoisomers” denotes stereoisomers for which conversion from        one to the other of these configurational stereoisomers requires        the cleavage/reformation of an interatomic covalent bond. Thus,        the term “configurational stereoisomers” denotes stereoisomers        which are not conformational isomers (or “rotamers”, for which        conversion from one to the other of the conformational isomers        is accompanied only by rotation of a part of the molecule about        the axis of a σ (sigma) bond formed by axial orbital overlap);    -   the term “hetero-stereoisomer” of difethialone denotes the        configurational stereoisomer of difethialone of formula        3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,        in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene        group of said hetero-stereoisomer have different absolute        configurations (i.e. 1S-3R and 1R-3S), the absolute        configurations being determined according to the sequential        priority rules and the Cahn-Ingold-Prelog (CIP) nomenclature;    -   the term “homo-stereoisomer” of difethialone denotes the        configurational stereoisomer of difethialone of formula 3-(4′        -bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,        in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene        group of said homo-stereoisomer have the same absolute        configuration (i.e. 1S-3S and 1R-3R);    -   the term “amount” means a molar amount, a mass amount or a        volume amount. The proportions are thus proportions of a molar        amount relative to a molar amount, of a mass amount relative to        a mass amount, or of a volume amount relative to a volume        amount;    -   the term “substantially” indicates, in the usual manner, that a        structural or functional characteristic should not be taken as        marking an abrupt discontinuity, which would have no physical        meaning, but covers not only this structure or this function,        but also slight variations of this structure or of this function        which produce, in the technical context under consideration, an        effect of the same nature, or else of the same degree;    -   the expressions “high-pressure liquid chromatography” or        “high-performance liquid chromatography” (HPLC) denote “HPLC”        chromatography or “High-Performance Liquid Chromatography”; and    -   the term “retention time” denotes the time, measured at the top        of the peak in the chromatogram, for which a compound is        retained on a chromatography column.

The invention relates to the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone. The invention relates to thedextrorotatory enantiomer of said homo-stereoisomer of difethialone inisolated form and in particular separated from the laevorotatoryenantiomer of said homo-stereoisomer of difethialone and from thelaevorotatory and dextrorotatory enantiomers of a configurationalstereoisomer of difethialone, named hetero-stereoisomer, in whichcarbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group havedifferent absolute configurations.

The inventors have discovered that it is possible to separate thelaevorotatory and dextrorotatory enantiomers of said homo-stereoisomerof difethialone and the laevorotatory and dextrorotatory enantiomers ofsaid hetero-stereoisomer of difethialone by high-pressure liquidchromatography in isocratic mode and under particular conditions byusing a chromatography column comprising a chiral stationary phase.Specifically, it was not known at the date of the invention how toseparate the configurational stereoisomers of difethialone andespecially the laevorotatory enantiomer and the dextrorotatoryenantiomer of said homo-stereoisomer of difethialone.

The inventors succeeded in performing this separation by choosing aparticular HPLC chromatography column, LUX® Cellulose-3 (Phenomenex, LePecq, France) of dimensions 150×2 mm and comprising a chiral stationaryphase constituted of porous particles of cellulose tris(4-methylbenzoate), having a particle size of 3 μm and a porosity of1000 Å. They used, as mobile phase, an eluent formed from a mixture ofacetonitrile (A) and water comprising formic acid in a volume proportionof 0.1% in water (B) with an A/B volume ratio of 80/20. The flow rate ofthe mobile phase in the column is maintained at a value of 0.25mL/minute and the separation is performed at a temperature of 23.2° C.The composition to be analysed is at a concentration of 1 μg ofdifethialone per millilitre in acetonitrile and the volume injected ontothe column is 1 μL. Detection may be performed by tandem massspectrometry (MS/MS). Detection may also be performed by photometry orby spectrophotometry by adjusting the difethialone concentration and theinjection volume for the purpose of obtaining optimum detection and bymeasuring the value of the area under the peak for each configurationalstereoisomer.

Under these experimental conditions, the value of the retention time(t₄) for the dextrorotatory enantiomer of said homo-stereoisomeraccording to the invention may vary according to the operatingconditions—especially according to the column temperature conditions—andmay be between 14.0 minutes and 14.4 minutes. The value of the retentiontime (t₁) for the laevorotatory enantiomer of said homo-stereoisomer mayvary according to the operating conditions—especially according to thecolumn temperature conditions—and may be between 7.8 minutes and 8.2minutes, such that the dextrorotatory and laevorotatory enantiomers ofsaid homo-stereoisomer may be separated by high-pressure liquidchromatography on a chiral column.

Under these same experimental conditions, the value of the retentiontime (t₃) for the dextrorotatory enantiomer of said hetero-stereoisomerof difethialone may vary according to the operatingconditions—especially according to the column temperature conditions—andmay be between 11.3 minutes and 11.8 minutes. The value of the retentiontime (t₂) for the laevorotatory enantiomer of said hetero-stereoisomerof difethialone may vary according to the operatingconditions—especially according to the column temperature conditions—andmay be between 9.0 minutes and 9.5 minutes.

Thus, under these analytical conditions, the order of elution of theconfigurational stereoisomers of difethialone is such that t₁<t₂<t₃<t₄.The retention time values t₁, t₂, t₃ and t₄ are liable to vary,especially with the temperature of the chromatography column. However,under these chromatographic conditions, the order of elution of theconfigurational stereoisomers of difethialone remains unchanged.

The invention thus relates to the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone in isolated form and having theproperty of being able to be eluted, under the chromatography conditionsdescribed above, last from among the four configurational stereoisomersof difethialone.

The dextrorotatory enantiomer of said homo-stereoisomer of difethialoneisolated in pure form according to the invention, dissolved in methanolat a concentration of 0.96 g/L and placed in a quartz spectrophotometrycuvette, has a circular dichroism spectrum acquired at 25° C. withpositive circular dichroism values between 220 nm and 300 nm.

The dextrorotatory enantiomer of said homo-stereoisomer of difethialoneisolated in pure form according to the invention, dissolved inchloroform (CHCl₃) has a specific optical rotation [α]²⁵° C._(589nm),measured at 25° C. and on the sodium D line (589 nm), having a value of+19.9°.

The dextrorotatory enantiomer of said homo-stereoisomer of difethialoneisolated in pure form according to the invention has, on proton magneticresonance (¹H-NMR) spectroscopy at 500 MHz in CDCl₃, a multiplet with achemical shift (6) of between 4.9 ppm and 5.1 ppm corresponding to theproton borne by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group ofsaid homo-stereoisomer of difethialone.

Said hetero-stereoisomer of difethialone and said homo-stereoisomer ofdifethialone are distinguished by their proton NMR spectra. In theproton NMR spectrum acquired in CDCl₃, the chemical shift of the protonborne by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group of saidhetero-stereoisomer of difethialone is about 5.3 ppm.

The invention also relates to a composition comprising thedextrorotatory enantiomer of said homo-stereoisomer of difethialoneaccording to the invention, with the exclusion of a racemic mixture ofdextrorotatory and laevorotatory enantiomers of said homo-stereoisomerof difethialone, i.e. of an optically inactive mixture of thedextrorotatory enantiomer of said homo-stereoisomer of difethialone andof the laevorotatory enantiomer of said homo-stereoisomer ofdifethialone.

The invention thus also relates to a composition comprising adextrorotatory enantiomer of the configurational stereoisomer ofdifethialone, named homo-stereoisomer, of formula3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group ofsaid homo-stereoisomer have the same absolute configuration, with theexclusion of a mixture in which the dextrorotatory enantiomer and thelaevorotatory enantiomer of said homo-stereoisomer of difethialone arein the same amount.

Advantageously and according to the invention, said homo-stereoisomer ispredominantly in dextrorotatory enantiomer form.

Throughout the text, the term “said homo-stereoisomer is predominantlyin dextrorotatory enantiomer form” means that the (mass, molar orvolume) amount of dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone is the major amount—greater than 50% —in all of saidhomo-stereoisomer of difethialone present in the composition (in all itsdextrorotatory and laevorotatory enantiomer forms), i.e. the (mass,molar or volume) amount of dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone in the composition is such that theratio of this amount to the (total) amount of said homo-stereoisomer inthe composition is greater than 50%.

In particular, in a composition according to the invention:

-   -   the ratio of the amount of dextrorotatory enantiomer of said        homo-stereoisomer of difethialone to the sum of the amounts of        each of the (dextrorotatory and laevorotatory) enantiomers of        said homo-stereoisomer of difethialone is greater than 0.5        (greater than 50%);    -   the ratio of the concentration of dextrorotatory enantiomer of        said homo-stereoisomer of difethialone to the sum of the        concentrations of each of the (dextrorotatory and laevorotatory)        enantiomers of said homo-stereoisomer of difethialone is greater        than 0.5 (greater than 50%); and    -   the proportion of dextrorotatory enantiomer of said        homo-stereoisomer of difethialone in the composition is greater        than the proportion of the laevorotatory enantiomer of said        homo-stereoisomer of difethialone.

Advantageously and according to the invention, the composition comprisesan amount of the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone in the composition such that the ratio of this amount tothe total amount of said homo-stereoisomer of difethialone in thecomposition is greater than 50%, especially greater than 60%, inparticular greater than 70%, more particularly greater than 80%,preferably greater than 90%, more preferentially greater than 95%,particularly preferentially greater than 98%, even more preferentiallygreater than 99% or about 100%. Advantageously, it comprises an amountof the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone in the composition such that the ratio of this amount tothe total amount of said homo-stereoisomer of difethialone in thecomposition is between 85% and 100%, more preferentially between 90% and98%. Advantageously, it comprises an amount of the dextrorotatoryenantiomer of said homo-stereoisomer of difethialone in the compositionsuch that the ratio of this amount to the total amount of saidhomo-stereoisomer of difethialone in the composition is between 98% and100%.

Advantageously, the composition may also comprise the laevorotatoryenantiomer of said homo-stereoisomer of difethialone, but in aproportion in the composition which is less than 50% —especially lessthan 25%, preferentially between 0% and 25%, in particular less than 10%—relative to the homo-stereoisomer of difethialone.

Advantageously and according to the invention, the difethialone ispredominantly in the dextrorotatory enantiomer form of saidhomo-stereoisomer of difethialone. The composition comprises an amountof the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone in the composition such that the ratio of this amount tothe amount of each of the enantiomers (of said hetero-stereoisomer andof said homo-stereoisomer) of difethialone is greater than 25%.

Advantageously, in a composition according to the invention:

-   -   the ratio of the amount of dextrorotatory enantiomer of said        homo-stereoisomer of difethialone to the sum of the amounts of        each of the enantiomers of said homo-stereoisomer of        difethialone and of each of the enantiomers of said        hetero-stereoisomer of difethialone is greater than 0.25        (greater than 25%);    -   the ratio of the concentration of dextrorotatory enantiomer of        said homo-stereoisomer of difethialone relative to the sum of        the concentrations of each of the enantiomers of said        homo-stereoisomer of difethialone and of each of the enantiomers        of said hetero-stereoisomer of difethialone is greater than 0.25        (greater than 25%); and    -   the proportion of dextrorotatory enantiomer of said        homo-stereoisomer of difethialone in the composition is greater        than the proportion of each of the enantiomers of said        homo-stereoisomer of difethialone and of said        hetero-stereoisomer of difethialone. In a composition according        to the invention, the proportion of dextrorotatory enantiomer of        said homo-stereoisomer of difethialone is greater than 25%        relative to the (total) difethialone.

Advantageously and according to the invention, the composition comprisesan amount of the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone in the composition such that the ratio of this amount tothe amount of total difethialone in the composition is greater than 25%,especially greater than 50%, in particular greater than 70%, moreparticularly greater than 80%, preferably greater than 90%, particularlypreferentially greater than 95%, more preferentially greater than 98%,even more preferentially greater than 99% or about 100%. A compositionaccording to the invention thus comprises difethialone predominantly indextrorotatory enantiomer form of said homo-stereoisomer ofdifethialone.

Advantageously, the composition comprises an amount of thedextrorotatory enantiomer of said homo-stereoisomer of difethialone suchthat the ratio of this amount to the amount of total difethialone isgreater than 70%, preferably between 80% and 100%, more preferentiallybetween 90% and 100%. Advantageously, the composition comprises anamount of the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone such that the ratio of this amount to the amount of totaldifethialone is between 95% and 99%. Advantageously, it comprises anamount of the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone such that the ratio of this amount to the amount of totaldifethialone is between 98% and 100%. Advantageously, the compositioncomprises an amount of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone such that the ratio of this amount tothe amount of total difethialone is about 100%.

Advantageously and according to the invention, the composition comprisesan amount of the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone in the composition such that the ratio of this amount tothe amount of total difethialone in the composition is greater than 97%.

A composition according to the invention may be substantially free oflaevorotatory enantiomer of said homo-stereoisomer of difethialone, i.e.the laevorotatory enantiomer of said homo-stereoisomer of difethialonemay be present in the composition, but only in trace amount. It may alsobe substantially free of said hetero-stereoisomer of difethialone, i.e.said hetero-stereoisomer of difethialone may be present in thecomposition, but only in trace amount.

Advantageously and according to the invention, the composition is inliquid form and comprises a liquid solvent for difethialone. It may be asolution of difethialone in a solvent for difethialone, with theexclusion of a racemic mixture of said laevorotatory and dextrorotatoryenantiomers of said homo-stereoisomer of difethialone. It may also be asolution comprising difethialone in a solvent for difethialone and inwhich said homo-stereoisomer of difethialone is predominantly indextrorotatory enantiomer form. It may also be a solution comprisingdifethialone in a solvent for difethialone and in which the difethialoneis predominantly in the dextrorotatory enantiomer form of saidhomo-stereoisomer of difethialone.

Advantageously and according to the invention, the composition is insolid form. It may also be a solid comprising difethialone, with theexclusion of a racemic mixture of the dextrorotatory and laevorotatoryenantiomers of said homo-stereoisomer of difethialone. It may also be asolid comprising difethialone and in which said homo-stereoisomer ofdifethialone is predominantly in dextrorotatory enantiomer form. It mayalso be a solid comprising difethialone and in which the difethialone ispredominantly in the dextrorotatory enantiomer form of saidhomo-stereoisomer of difethialone.

The invention thus also relates to a composition comprising thedextrorotatory enantiomer according to the invention, with the exclusionof a racemic mixture of the laevorotatory and dextrorotatory enantiomersof said homo-stereoisomer of difethialone, the difethialone of thecomposition being optically active and dextrorotatory. However, it isnot excluded for the composition according to the invention, comprisingthe dextrorotatory enantiomer according to the invention with theexclusion of a racemic mixture of the laevorotatory and dextrorotatoryenantiomers of said homo-stereoisomer of difethialone, to itself beoptically active and laevorotatory, or alternatively optically inactive.

The invention also relates to the use of a composition according to theinvention for the preparation of a rodenticidal bait for target rodentpests.

The invention also relates to a rodenticidal bait comprising acomposition according to the invention, and at least one excipient thatis edible for target rodent pests.

A rodenticidal bait according to the invention comprises:

-   -   at least one excipient that is edible for target rodent pests,    -   the dextrorotatory enantiomer of a configurational stereoisomer        of difethialone, named homo-stereoisomer, of formula        3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,        in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene        group of said homo-stereoisomer have the same absolute        configuration, with the exclusion of a racemic mixture of        dextrorotatory and laevorotatory enantiomers of said        homo-stereoisomer of difethialone. In a bait according to the        invention, the dextrorotatory enantiomer of said        homo-stereoisomer of difethialone and the laevorotatory        enantiomer of said homo-stereoisomer of difethialone are in        different amounts.

Advantageously, a bait according to the invention comprises an excipientthat is edible for target rodent pests and said homo-stereoisomer ofdifethialone predominantly in dextrorotatory enantiomer form.

The inventors, who succeeded in separating the dextrorotatory enantiomerfrom the laevorotatory enantiomer of said homo-stereoisomer ofdifethialone and from said hetero-stereoisomer of difethialone and inisolating the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone, also observed, entirely surprisingly, that thedextrorotatory enantiomer of said homo-stereoisomer of difethialone andthe laevorotatory enantiomer of said homo-stereoisomer of difethialonedo not have the same persistence in the liver of target rodent pests andthat the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone is in fact the enantiomer of said homo-stereoisomer ofdifethialone that is the less persistent in the liver of target rodents,especially of target rodent pests.

They observed that the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone, which is less persistent anddisappears from the liver of the target rodent pest which has consumed abait according to the invention more rapidly than the disappearance ofthe laevorotatory enantiomer of said homo-stereoisomer of difethialone,is, however, rodenticidal.

The residual dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone in the body of a poisoned rodent disappears more quicklythan the other (laevorotatory) enantiomer of said homo-stereoisomer ofdifethialone. The dead or live target rodent pest which has ingested thedextrorotatory enantiomer of said homo-stereoisomer of difethialone isthus sparingly toxic with respect to non-rodent mammals and birds whichconsume the target rodent pest—dead or alive—and in particular withrespect to predators (especially non-rodent mammals and birds) whichpreferentially consume the viscera of their prey and in particular theirliver.

The inventors have also observed that the dextrorotatory enantiomer ofsaid homo-stereoisomer of difethialone, although having low hepaticpersistence in target rodent pests, does in fact make it possible, in anentirely surprising manner to date, to efficiently control target rodentpests.

Advantageously and according to the invention, the rodenticidal baitcomprises a mass amount of difethialone such that the ratio (massproportion) of this mass amount of difethialone to the mass amount ofrodenticidal bait is less than 200 ppm, i.e. less than 200 mg ofdifethialone per kilogram of bait. Advantageously, the mass proportionof difethialone in the rodenticidal bait is between 1 ppm and 100 ppm (1mg to 100 mg of difethialone per kilogram of rodenticidal bait),especially between 5 ppm and 100 ppm (5 mg to 100 mg of difethialone perkilogram of rodenticidal bait), preferably between 5 ppm and 50 ppm (5mg to 50 mg of difethialone per kilogram of rodenticidal bait), morepreferentially between 10 ppm and 50 ppm (10 mg to 50 mg of difethialoneper kilogram of rodenticidal bait), even more preferentially between 15ppm and 50 ppm (15 mg to 50 mg of difethialone per kilogram ofrodenticidal bait), for example about 15 ppm (15 mg of difethialone perkilogram of bait).

Advantageously and according to the invention, the edible excipientcomprises at least one food chosen from the group formed from cerealseeds, especially hulled cereal seeds, cereal seed meals, cereal seedflours, cereal seed flakes, cereal bran and non-cereal seeds, forexample alfalfa seeds, especially in hulled form, in the form of meal,in the form of flour, or in the form of flakes or bran. The edibleexcipient may comprise any support that can be consumed by target rodentpests.

Advantageously, the edible excipient comprises at least one food chosenfrom the group formed from foods of plant origin and foods of animalorigin. Advantageously, the edible excipient comprises at least one foodchosen to stimulate the appetite of the target rodent pests. Inparticular, this food is chosen from the group formed from seeds of oneor more cereals, hulled seeds of one or more cereals, meals of seeds ofone or more cereals, flakes of seeds of one or more cereals, bran of oneor more cereals and flour of seeds of one or more cereals. By way ofexample, the cereals are chosen from the group formed from oat, wheat,barley, corn, soybean and rice.

Advantageously, the food is chosen from the group formed from sweetenedfoods. For example, they may be foods comprising at least one sugarchosen from the group formed from sucrose, lactose, fructose andglucose. It may be a sugar syrup—for example a sugar syrup obtained byhydrolysis of starch—or a sugar syrup obtained by hydrolysis of sucrose(invert sugar syrup), or a beet sugar syrup, or a maple syrup or asugarcane syrup, or a syrup obtained from a plant of the Stevia genus.

Advantageously, the food is chosen from the group formed from coconutalbumen (copra) flakes and flour. Advantageously, the food is chosenfrom the group formed from walnuts, hazelnuts and almonds—in gratedand/or powder form.

Advantageously, the food is chosen from the group formed from plantfats, plant oils (for example rapeseed oil, soybean fat, sunflower oil,cocoa butter, groundnut oil, groundnut butter, corn oil, palm oil),animal fats and animal oils (butter, lard, fish oil).

Advantageously, the food is chosen from the group formed from proteinsof plant origin and proteins of animal origin. By way of example,examples that may be mentioned include powdered milk, especiallypowdered skimmed milk, eggs, especially powdered eggs, proteinhydrolysates of animal origin and protein hydrolysates of plant origin.

The excipient that is edible for target rodent pests is chosen to allowconsumption of the bait by target rodent pests. Advantageously andaccording to the invention, each edible excipient is non-lethal totarget rodent pests. The edible excipient is not in itself rodenticidal.

Advantageously and according to the invention, the rodenticidal bait ischosen from the group formed from solid baits comprising difethialoneand a solid edible excipient. Advantageously, the rodenticidal bait is asolid in divided form, for example in the form of balls or granules.Advantageously, the rodenticidal bait may be a solid in block or pasteform that may be consumed by the target rodent pests or a solid materialthat may be nibbled by the target rodent pests. Advantageously, thesolid rodenticidal bait according to the invention may be in the form ofa rigid block, a semi-rigid block, a foam, a powder or a gel.

Advantageously, the rodenticidal bait which is in the form of a powder,in the form of a foam or in the form of a gel is suitable for soilingthe fur of the target rodent pest(s) and for being ingested by saidpest(s) during their grooming.

It may be a solid rodenticidal bait comprising difethialone, with theexclusion of a racemic mixture of the dextrorotatory and laevorotatoryenantiomers of said homo-stereoisomer of difethialone. It may also be asolid rodenticidal bait comprising difethialone and in which saidhomo-stereoisomer of difethialone is predominantly in dextrorotatoryenantiomer form. It may also be a solid rodenticidal bait comprisingdifethialone and in which the difethialone is predominantly in thedextrorotatory enantiomer form of said homo-stereoisomer ofdifethialone.

Advantageously and according to the invention, the rodenticidal bait ischosen from the group formed from liquid baits comprising difethialoneand a liquid edible excipient. The rodenticidal bait is then a drink fortarget rodent pests.

Advantageously and according to the invention, the rodenticidal bait ischosen from the group formed from liquid baits comprising difethialoneand a liquid edible excipient. The rodenticidal bait is then a drink fortarget rodent pests. It may be a solution of difethialone in a solventfor difethialone, with the exclusion of a racemic mixture ofdextrorotatory and laevorotatory enantiomers of said homo-stereoisomerof difethialone. It may also be a solution of difethialone in a solventfor difethialone and in which said homo-stereoisomer of difethialone ispredominantly in dextrorotatory enantiomer form. It may also be asolution of difethialone in a solvent for difethialone and in which thedifethialone is predominantly in the dextrorotatory enantiomer form ofsaid homo-stereoisomer of difethialone. It may also be a suspension oran emulsion of difethialone in a liquid medium.

The invention thus also relates to a rodenticidal bait in which thedifethialone is optically active. However, it is not excluded for thedifethialone of the rodenticidal bait according to the invention to beoptically inactive.

Advantageously, the rodenticidal bait comprises at least one dye. Such adye makes it possible in particular to give said rodenticidal bait acolour that is readily detectable and identifiable by a person handlingthe rodenticidal bait.

Advantageously, the rodenticidal bait comprises at least one preservingagent capable of ensuring its conservation during its storage.Advantageously, the rodenticidal bait comprises at least one bitteringcompound such as denatonium benzoate, also known as Bitrex®, which isintended to reduce the risks of accidental consumption by non-targetorganisms.

Advantageously, in one particular variant, the composition and therodenticidal bait according to the invention exclusively comprisedifethialone—in which the homo-stereoisomer is not a racemic mixture—asrodenticidal substance. In particular, the composition and therodenticidal bait according to the invention are free of any otheranticoagulant substance for rodenticidal use. However, in this variantaccording to the invention, the composition and the rodenticidal baitmay comprise any pest-control substance other than a rodenticide, suchas an insecticidal and/or acaricidal substance.

Advantageously, in another particular variant, the composition and therodenticidal bait according to the invention comprise difethialone withthe exclusion of a racemic mixture of laevorotatory and dextrorotatoryenantiomers of said homo-stereoisomer and at least one other substancedifferent from difethialone as rodenticidal substance. This otherrodenticidal substance different from difethialone may be anotheranticoagulant substance—especially of the anti-vitamin K type or not—oranother non-anticoagulant rodenticidal substance.

The invention also relates to a process for controlling target rodentpests, in which there is spread an amount of rodenticidal baitcomprising:

-   -   at least one excipient that is edible for target rodent pests;        and    -   the dextrorotatory enantiomer of the configurational        stereoisomer of difethialone, named homo-stereoisomer, the        formula of which is        3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,        in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene        group of said homo-stereoisomer have the same absolute        configuration;        with the exclusion of a racemic mixture of the dextrorotatory        and laevorotatory enantiomers of said homo-stereoisomer of        difethialone.

The invention also relates to a process for controlling target rodentpests, in which there is spread an amount of rodenticidal bait accordingto the invention, said amount of bait being sufficient to berodenticidal.

An amount of rodenticidal bait comprising said homo-stereoisomer ofdifethialone predominantly in dextrorotatory enantiomer form, which hasreduced hepatic persistence in the target rodent pest and, surprisingly,rodenticidal efficacy that is at least maintained, is thus spread. Theprocess according to the invention thus makes it possible to limit thesecondary poisoning of non-rodent mammals and birds that are liable tofeed on poisoned rodents which are dead or alive but comprising areduced amount—and especially a non-lethal amount—of difethialone. Theprocess according to the invention also makes it possible to limit suchsecondary poisoning of non-rodent mammals and birds that are liable topreferentially consume the viscera—in particular the liver—of said deador live poisoned rodents.

Advantageously and according to the invention, said homo-stereoisomer ofdifethialone is predominantly in dextrorotatory enantiomer form.Advantageously and according to the invention, the difethialone ispredominantly in the dextrorotatory enantiomer form of saidhomo-stereoisomer of difethialone.

Advantageously and as a variant according to the invention, thefollowing are chosen in combination:

-   -   the edible excipient;    -   a proportion of dextrorotatory enantiomer of said        homo-stereoisomer of difethialone relative to the        homo-stereoisomer of difethialone;    -   a proportion of dextrorotatory enantiomer of said        homo-stereoisomer of difethialone relative to the difethialone;    -   a mass proportion of difethialone relative to the rodenticidal        bait; and    -   an amount of spread bait;        so that target rodent pests consume an amount of difethialone        that is sufficient to be lethal to said target rodent pests        which consume said bait in the course of a single period of 24        consecutive hours.

A rodenticidal bait according to this variant of the invention is a baitthat is mortal in a single intake, or a “one-shot” bait. Advantageouslyand according to this variant of the invention, the mass proportion ofdifethialone in the rodenticidal bait is between 2 ppm and 200 ppm,especially between 5 ppm and 100 ppm, preferably between 10 ppm and 50ppm, more preferentially between 15 ppm and 50 ppm.

Advantageously and in another variant according to the invention, thefollowing are chosen in combination:

-   -   the edible excipient;    -   a proportion of dextrorotatory enantiomer of said        homo-stereoisomer of difethialone relative to the        homo-stereoisomer of difethialone;    -   a mass proportion of difethialone relative to the rodenticidal        bait; and    -   an amount of spread bait;        so that target rodent pests consume an amount of difethialone:    -   which is non-lethal to target rodent pests, i.e. which is        generally non-lethal to target rodent pests which consume said        bait over a period of 24 consecutive hours; and    -   which is sufficient to be lethal to target rodent pests which        consume said bait over several 24-hour periods.

Advantageously, the 24-hour periods are consecutive.

This other variant of the invention is thus also directed towards aprocess for controlling target rodent pests, in which there is spread anamount of rodenticidal bait that is lethal to target rodent pestsdurably consuming this rodenticidal bait and non-lethal to non-targetrodents or animals accidentally consuming this rodenticidal bait. Thisis then referred to as a “multi-dose” or “multi-feeding” controlprocess. In such a process according to the invention, the consumptionof rodenticidal bait by a target rodent pest over a period of 24 hoursis generally insufficient to result in the death of said rodent, whereasrepeated consumption of rodenticidal bait over at least two days resultsin the death of the target rodent pest.

The invention is thus directed towards a process for controlling apopulation of target rodent pests, in which target rodent pests areprovided with an amount of rodenticidal bait that is liable to beingested by the target rodent pests, said amount of rodenticidal baitbeing sufficient to kill target rodent pests which consume saidrodenticidal bait over several days.

Advantageously, in this other variant of the invention, an amount ofrodenticidal bait is spread so that target rodent pests consume anamount of difethialone that is sufficient to be lethal to said targetrodent pests which consume said bait over several 24-hour periods, saidperiods being consecutive.

Advantageously and in this other variant according to the invention, themass proportion of difethialone is between 5 ppm and 50 ppm, especiallybetween 5 ppm and 30 ppm—especially about 15 ppm—relative to therodenticidal bait.

Advantageously, the amount of rodenticidal bait spread, the massproportion of difethialone relative to the rodenticidal bait and theproportion of dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone relative to the difethialone are adapted so that theconsumption of the rodenticidal bait is lethal to target rodent pestswhich daily consume bait over at least two 24-hour periods, especiallyfrom 3 to 7 periods, at least two—and especially all—of the periodsbeing consecutive.

Advantageously, in this other variant of a process according to theinvention, since the proportion of dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone is greater than 95%—especially about100%—relative to the difethialone, the mass proportion of difethialonerelative to the rodenticidal bait is between 5 ppm and 50 ppm,especially about 15 ppm.

In a process according to the invention, an amount of rodenticidal baitthat is sufficient to satisfy their daily appetite is provided to targetrodent pests, said rodenticidal bait comprising a major proportion ofdextrorotatory enantiomer of said homo-stereoisomer of difethialone.

In a process according to the invention, the amount of rodenticidal baitspread, the proportion of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone relative to the difethialone and themass proportion of difethialone relative to the rodenticidal bait areadapted so as to allow consumption of rodenticidal bait for several daysby target rodent pests, while at the same time limiting:

-   -   the risks of primary intoxication of non-target mammals and        birds which are liable to consume such a rodenticidal bait only        occasionally and accidentally;    -   the risks of secondary intoxication, for example of predators of        target rodents, which are liable to consume target rodents—dead        or live—that have ingested an amount of said bait.

Advantageously and according to the invention, the amount of baitspread, the mass proportion of difethialone relative to the bait and theproportion of dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone are adapted to limit the (total) amount of difethialone inthe liver of rodents which have died by consuming said bait.

The invention also relates to a chromatographic process for obtaining adextrorotatory enantiomer of a configurational stereoisomer ofdifethialone, named homo-stereoisomer, the formula of which is3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group ofsaid homo-stereoisomer have the same absolute configuration, in whichprocess:

-   -   a high-pressure liquid chromatography column of dimensions 150×2        mm, and comprising a chiral stationary phase constituted of        cellulose tris (4-methylbenzoate) particles, said particles        having a mean size of 3μm and having a mean pore size of 1000 Å,        is chosen;    -   a mixture formed from acetonitrile (A) and water comprising 0.1%        by volume of formic acid (B), with an A/B volume ratio of 80/20        and with a flow rate of the liquid mobile phase in the        chromatography column of 0.25 mL/minute, is chosen as liquid        mobile phase;    -   separation of the configurational stereoisomers of difethialone        is performed at room temperature, during which:    -   a liquid composition comprising said dextrorotatory enantiomer        of said homo-stereoisomer of difethialone is introduced into the        top of the chromatography column; and then    -   the liquid composition is entrained with the mobile phase in the        chromatography column under conditions suitable for separating        the configurational stereoisomers of difethialone; and    -   a fraction of the mobile phase comprising said dextrorotatory        enantiomer of said homo-stereoisomer of difethialone is        collected with a retention time t₄ having a value such that        t₁<t₂<t₃<t₄; t₁, t₂ and t₃ representing the retention times of        each of the configurational stereoisomers of difethialone        different from the dextrorotatory enantiomer of said        homo-stereoisomer of difethialone, separately from a        laevorotatory enantiomer of said homo-stereoisomer of        difethialone with a retention time t₁ and separately from the        laevorotatory and dextrorotatory enantiomers of a        configurational stereoisomer of difethialone, named        hetero-stereoisomer, in which carbons 1 and 3 of the        1,2,3,4-tetrahydronaphthalene group of said hetero-stereoisomer        have different absolute configurations, and of retention times        t₂ and t₃; and then    -   the liquid mobile phase of said fraction is removed so as to        obtain said dextrorotatory enantiomer of said homo-stereoisomer        of difethialone.

The invention also relates to said dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone obtained via a process according tothe invention.

The invention also relates to a configurational stereoisomer ofdifethialone, to a process for obtaining such a configurationalstereoisomer, to a composition comprising such a configurationalstereoisomer, to a rodenticidal bait and to a process for controllingtarget rodent pests, characterized in combination by all or some of thecharacteristics mentioned hereinabove or hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

Other aims, characteristics and advantages of the invention will emergeon reading the following description and the examples, which are givenfor purely non-limiting purposes and which refer to the attachedfigures, in which:

FIG. 1 is a chromatogram of an analysis by high-pressure liquidchromatography on a chiral column of difethialone (top) and of thedextrorotatory enantiomer of said purified homo-stereoisomer ofdifethialone (bottom);

FIG. 2 is a proton NMR spectrum at 300 MHz of said homo-stereoisomer ofdifethialone;

FIG. 3 is a proton NMR spectrum at 300 MHz of said hetero-stereoisomerof difethialone;

FIG. 4 is a proton NMR spectrum at 500 MHz of the dextrorotatoryenantiomer of said homo-stereoisomer of difethialone according to theinvention;

FIG. 5 is a ¹³C carbon NMR spectrum at 500 MHz of the dextrorotatoryenantiomer of said homo-stereoisomer of difethialone;

FIG. 6 is an analysis by proton NMR ('H-NMR) correlation spectroscopy at500 MHz of the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone;

FIG. 7 is a circular dichroism spectrum of the dextrorotatory enantiomerof said homo-stereoisomer of difethialone;

FIG. 8 is a representation in graph form of the change over time of thehepatic concentration in rats (male and female) of the dextrorotatoryenantiomer of said homo-stereoisomer of difethialone (●) and of thelaevorotatory enantiomer of said homo-stereoisomer of difethialone (◯);

FIG. 9 is a representation in graph form of the change over time of thehepatic concentration in rats (male and female) of the dextrorotatoryenantiomer of said homo-stereoisomer of difethialone (●), of saidhomo-stereoisomer of difethialone (□) and of said hetero-stereoisomer ofdifethialone (▪); and

FIG. 10 is a representation in graph form of the change over time of themean hepatic concentration in rats of the dextrorotatory enantiomer ofsaid homo-stereoisomer of difethialone (●) and of the total difethialone(Δ).

DETAILED DESCRIPTION OF THE INVENTION

A. Purification of the Dextrorotatory Enantiomer of said IsolatedHomo-Stereoisomer of DifethialoneA.1. Identification of Said Homo-Stereoisomer of Difethialone and ofsaid Hetero-Stereoisomer of Difethialone

The homo-stereoisomer of difethialone is identified by proton magneticresonance (¹H-NMR) spectroscopy. The homo-stereoisomer of difethialonedissolved in CDCl₃ has a multiplet with a chemical shift (δ) of between4.9 ppm and 5.1 ppm and corresponding to the proton borne by carbon 1 ofthe 1,2,3,4-tetrahydronaphthalene group of difethialone as illustratedin FIG. 2.

It is possible to distinguish the hetero-stereoisomer of difethialoneand the homo-stereoisomer of difethialone by their proton NMR spectra.In the proton NMR spectrum acquired in CDCl₃, the chemical shift of theproton borne by carbon 1 of the 1,2,3,4-tetrahydronaphthalene group ofsaid hetero-stereoisomer of difethialone (FIG. 3) is between 5.2 ppm and5.4 ppm.

A.2. Separation of the Laevorotatory and Dextrorotatory Enantiomers ofsaid Homo-Stereoisomer of Difethialone by High-Pressure LiquidChromatography on a Chiral Column

The inventors solved the complex and hitherto unresolved problem ofseparating the configurational isomers of difethialone and in particularthe laevorotatory and dextrorotatory enantiomers of saidhomo-stereoisomer of difethialone. They succeeded in performingseparation of the enantiomers of said homo-stereoisomer of difethialoneby high-pressure (high-performance) liquid chromatography on a LUX®Cellulose-3 column (Phenomenex, Le Pecq, France) of dimensions 150×2 mmand comprising a chiral stationary phase constituted of porous particlesof cellulose tris (4-methylbenzoate), with a particle size of 3 μm and aporosity of 1000 Å and using, as mobile phase, a mixture formed fromacetonitrile (A) and water comprising formic acid in a volume proportionof 0.1% (B), the AB volume proportion being 80/20. The flow rate of themobile phase in the column is 0.25 mL/minute and the separation isperformed at a temperature of 23.2° C. The solution containing thesample to be analysed is at a concentration of 1 μg of difethialone permillilitre in acetonitrile and is filtered through a regeneratedcellulose membrane with a cut-off threshold of 0.2 μm. The volume ofsolution containing the sample to be analysed injected onto the columnis 1 μL.

In a process for separating the enantiomers of said homo-stereoisomer ofdifethialone, it is possible to detect said enantiomers leaving thehigh-pressure liquid chromatography column by tandem mass spectrometry(MS/MS) in negative electrospray ionization mode (ESI: ElectroSprayIonization). The temperature of the nebulizer gas is 350° C. and itsflow rate is 8 L/minute. The pressure of the nebulizer gas is brought to2700 hPa. In particular, the MRM (“Multiple Reaction Monitoring”)transitions m/z 537.1→151.0 and m/z 537.1→78.9, corresponding to thedifethialone signals, are detected. FIG. 1 represents the chromatogramsof difethialone (top) and of the dextrorotatory enantiomer of theisolated homo-stereoisomer of difethialone (bottom).

Under these experimental conditions, the value of the retention time(t₄) for said dextrorotatory enantiomer of said homo-stereoisomeraccording to the invention is about 14.4 minutes as described in FIG. 1.By way of comparison, the value of the retention time (t₁) for thelaevorotatory enantiomer of said homo-stereoisomer is about 8.1 minutesas described in FIG. 1, and as such the dextrorotatory and laevorotatoryenantiomers of said homo-stereoisomer may be efficiently separated byhigh-pressure liquid chromatography on a chiral column.

The value of the retention time (t₃) for the dextrorotatory enantiomerof said hetero-stereoisomer of difethialone is about 11.7 minutes andthe value of the retention time (t₂) for the laevorotatory enantiomer ofsaid hetero-stereoisomer of difethialone is about 9.4 minutes. Thus,under these experimental conditions, the order of elution of theconfigurational stereoisomers of difethialone is such that t₁<t₂<t₃<t₄.

It is possible under these experimental conditions (stationary phase,mobile phase, temperature) to perform a preparative separation of thelaevorotatory and dextrorotatory enantiomers of said homo-stereoisomerof difethialone by using a similar stationary phase with a particle sizeof greater than 3 μm, and a chromatography column of larger dimensions,especially a diameter of 20 mm.

B. Structural Characterization of the Dextrorotatory Enantiomer of saidHomo-Stereoisomer of DifethialoneB.1. UV spectroscopy

The UV spectrum of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone dissolved in chloroform (CHCl₃) showsabsorbance peaks centred at 238.2 nm and 259.5 nm.

B.2. Optical Rotation

The inventors characterized the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone in isolated form by means of itsoptical rotation (also known as the optical activity or circularbirefringence), i.e. its ability to deviate the polarization plane ofpolarized light. Deviation of the polarization plane of polarized lightclockwise facing the polarized light beam characterizes a dextrorotatorysolution, and deviation of the polarization plane of polarized lightanticlockwise facing the polarized light beam characterizes alaevorotatory solution and compound.

The optical rotation of a solution of dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone in chloroform (CHCl₃) is measured at aconcentration of 11.6 g/L. The optical rotation of this solution ismeasured by means of a P 2000 digital polarimeter (JASCO, Bouguenais,France) operating with excitatory light with a wavelength of 589 nm. Themean optical rotation a obtained on two series of ten measurements is2.308°. The specific optical rotation at 25° C. [α]²⁵° C._(589nm) forthe dextrorotatory enantiomer of said homo-stereoisomer of difethialonedissolved in chloroform, measured on the sodium D line (589 nm), is+19.9°.

B.3. Circular dichroism

The circular dichroism spectrum of the isolated dextrorotatoryenantiomer of said homo-stereoisomer of difethialone reflects thedifference in absorbance (ΔA=A_(L)−A_(R)) of the two waves of leftcircular polarization (LCP) of intensity A_(L) and of right circularpolarization (RCP) of intensity A_(R). This makes it possible todistinguish the dextrorotatory and laevorotatory enantiomers of saidhomo-stereoisomer of difethialone. This difference in absorbance of thetwo circularly polarized waves is measured in a J-815 circular dichroismspectrometer (JASCO, Bouguenais, France). 2 mL of a solution ofdextrorotatory enantiomer of said homo-stereoisomer of difethialone inmethanol (CH₃OH) at a concentration of 0.96 mg/mL are prepared. Thesolution is transferred into a quartz spectrophotometer cuvette. Thecircular dichroism spectrum of the solution is measured at 25° C.between 210 nm and 400 nm. The circular dichroism spectrum of thedextrorotatory enantiomer of said homo-stereoisomer of difethialonemeasured under these conditions is shown in FIG. 7. The circulardichroism value is positive between the wavelengths of between 220 nmand 300 nm.

B.4. Nuclear Magnetic Resonance

FIGS. 2 and 3 represent, respectively, a proton nuclear magneticresonance spectrum (¹H-NMR) at 300 MHz of the homo-stereoisomer ofdifethialone in CDCl₃ (FIG. 2) and a proton nuclear magnetic resonancespectrum at 300 MHz in CDCl₃ of the hetero-stereoisomer of difethialonein CDCl₃ (FIG. 3). Said homo-stereoisomer of difethialone has (FIG. 2) amultiplet whose chemical shift (δ) is between 4.9 ppm and 5.1 ppm,corresponding to carbon 1 of the 1,2,3,4-tetrahydronaphthalene group ofsaid homo-stereoisomer of difethialone. Said hetero-stereoisomer ofdifethialone has (FIG. 3) a multiplet whose chemical shift (δ) isbetween 5.2 ppm and 5.4 ppm.

FIG. 4 is a proton NMR spectrum at 500 MHz of the dextrorotatoryenantiomer of said homo-stereoisomer of difethialone in CDCl₃. Theproton NMR spectra of the dextrorotatory and laevorotatory enantiomersof said homo-stereoisomer of difethialone are indistinguishable fromeach other.

FIG. 6 is a two-dimensional proton NMR (2D ¹H-NMR) spectrum obtained bycorrelation spectroscopy of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone dissolved in CDCl₃ at a concentrationof 40 mg/mL acquired on a Brüker Avance III HD spectrometer (500 MHz)equipped with a Prodigy motorized multi-core direct cryoprobe. It allowsidentification of the coupling of the proton borne by carbon 1 of the1,2,3,4-tetrahydronaphthalene group of said homo-stereoisomer ofdifethialone (5.1 ppm) with the protons borne by carbon 2 of the1,2,3,4-tetrahydronaphthalene group at 2.4 ppm.

FIG. 5 is a ¹³C NMR spectrum of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone dissolved in CDCl₃ at a concentrationof 40 mg/mL acquired on a Briiker Avance III HD spectrometer (500 MHz)equipped with a Prodigy motorized multi-core direct cryoprobe. It allowsidentification of the 31 carbon atoms of difethialone. The ¹³C-NMRspectrum of the dextrorotatory enantiomer of said homo-stereoisomer ofdifethialone is not distinguished from the ¹³C-NMR spectrum of thelaevorotatory enantiomer of said homo-stereoisomer of difethialone.However, said homo-stereoisomer of difethialone has a characteristicsignal between 34 ppm and 38 ppm which is distinctive for saidhetero-stereoisomer of difethialone.

C. Extraction of Difethialone from the Liver of Rats Treated withDifethialone for the Purpose of Analysis of the ConfigurationalStereoisomers of Difethialone

C.1. Homogenization of the Liver Sample

About 0.525 g (±0.025 g) of rat liver is weighed out accurately andplaced in a 50 mL polypropylene tube. 10 mL of acetone are added and thesuspension is homogenized using an Ultra-Turrax® homogenizer/disperserfor a time of about 30 seconds. The homogenizer/disperser shaft isrinsed with hot water and then twice with 20 mL of acetone in apolypropylene tube. The homogenate is centrifuged for 5 minutes at acentrifugation speed of 3000 rpm (revolutions per minute). Thesupernatant is collected and transferred into a test tube. The sample issubjected to evaporation under a stream of nitrogen (N₂) at atemperature of 40° C. so as to form a dry extract.

C.2. Lipid Removal

1 mL of acetonitrile is added to the tube containing the dry extract soas to dissolve it. The acetonitrile solution is washed twicesuccessively with 1 mL of hexane. The lipid-free extract is dried undera stream of nitrogen (N₂) at a temperature of 40° C. and is then takenup in 0.5 mL of methanol and dissolved by vortex stirring. 0.5 mL ofultra-pure (Milli-Q) water is then added. The sample isvortex-homogenized.

C.3. Solid-Phase Extraction (SPE) of Difethialone

1 mL of dichloromethane (CH₂Cl₂), then 1 mL of methanol (CH₃OH), then 1mL of ultra-pure (Milli-Q) water are passed through an Oasis HLB 1 cccartridge (WAT094225, Waters). The lipid-free liver extract (1 mLMeOH/Milli-Q H₂O) containing difethialone is then loaded onto the top ofthe preconditioned cartridge. The liver extract penetrates through thecartridge by gravity on contact with the solid phase of the cartridge. 1mL of washing solution formed from methanol and ultra-pure water in a90/10 volume proportion is then loaded onto the top of the cartridge.The cartridge is dried by suction under vacuum connected to the bottomof the cartridge. 1 mL of eluting solution formed from dichloromethaneand methanol in a 90/10 volume proportion is then loaded onto the top ofthe cartridge and an eluate comprising difethialone is collected at thebottom of the cartridge. The solvent of the eluate is evaporated offunder a stream of nitrogen (N₂) at a temperature of 40° C. The sample istaken up in 0.5 mL of acetonitrile and the acetonitrile solutioncontaining difethialone is filtered through a 0.2 μm filter.

C.4. Analysis

The acetonitrile solution containing difethialone is analysed byhigh-pressure liquid chromatography on a LUX® Cellulose-3 chiral column(Phenomenex, Le Pecq, France) (150×2 mm, particle size of 3 μm) asdescribed in point A2) above.

D. Study of the Hepatic Persistence of the Configurational Stereoisomersof Difethialone in Rats

A solution of a mixture of homo-stereoisomer (DFN-Homo-SI) and ofhetero-stereoisomer (DFN-Hetero-SI) of difethialone in a mixture ofvegetable oil and 5% DMSO is administered by tube-feeding (“per os”) to8-week-old coumaphen-sensitive rats (male and female Rattus norvegicus)weighing about 200 g. The molar proportion of homo-stereoisomer is 40%and the molar proportion of hetero-stereoisomer is 60%. Eachconfigurational stereoisomer of difethialone is formed from a racemicmixture of the two enantiomers of said corresponding configurationalstereoisomer.

On D0, a tube-feeding solution is administered to the rats so that theamount of difethialone ingested by each rat is about 3.4 mg per kilogramof rat. To avoid haemorrhage, the tube-fed rats are treated daily bysubcutaneous administration of a dose of vitamin K1 (as haemorrhageantidote) at a rate of 0.1 U per 200 g of live rat weight.

At 4 hours (H+4), 9 hours (H+9), 24 hours (H+24), 120 hours (H+120), 168hours (H+168) and 216 hours (H+216) after tube-feeding, three male ratsand three female rats anaesthetized beforehand with isoflurane areeuthanized, the liver of the euthanized rats is removed, thedifethialone is then extracted from the liver and the amount of each ofthe configurational stereoisomers of difethialone is assayed, the areaunder the peaks in the chromatogram obtained is measured and eachconfigurational stereoisomer is quantified by comparison with acalibration curve. The following are assayed:

-   -   the dextrorotatory enantiomer of said homo-stereoisomer;    -   the laevorotatory enantiomer of said homo-stereoisomer;    -   the dextrorotatory enantiomer of said hetero-stereoisomer;    -   the laevorotatory enantiomer of said hetero-stereoisomer;        present in the liver of the tube-fed rats.

The results are shown in FIGS. 8, 9 and 10, in which the content of saiddextrorotatory enantiomer of said homo-stereoisomer of difethialoneexpressed in nanograms of enantiomer per gram of liver (ng/g) is givenas a function of the time (in hours) after tube-feeding of the rats.

In FIG. 8, the content of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone in the liver (mean of the contentsmeasured on six rats expressed in nanograms of said enantiomer per gramof liver (ng/g)) is represented by filled circles (●) and the content ofthe laevorotatory enantiomer of said homo-stereoisomer of difethialonein the liver (mean of the contents measured on six rats expressed innanograms of said enantiomer per gram of liver (ng/g)) is represented byempty circles (◯). The hepatic content of the dextrorotatory enantiomerof said homo-stereoisomer of difethialone decreases more rapidly thanthe hepatic content of the laevorotatory enantiomer of saidhomo-stereoisomer of difethialone, whereas the initial proportion of thetwo enantiomers ingested by the rats is identical (20%). The values forthe measured hepatic contents are given in table 1 below, in which“DFN-Homo-SI” represents said homo-stereoisomer of difethialone,“DFN-Homo-dextro” represents the dextrorotatory enantiomer of thehomo-stereoisomer of difethialone according to the invention,“DFN-Homo-laevo” represents the laevorotatory enantiomer of thehomo-stereoisomer of difethialone, “DFN-Hetero-SI” represents saidhetero-stereoisomer of difethialone, “DFN-Hetero-laevo” represents thelaevorotatory enantiomer of the hetero-stereoisomer of difethialone and“DFN-Hetero-dextro” represents the dextrorotatory enantiomer of thehetero-stereoisomer of difethialone.

TABLE 1 Hepatic content, ng/g Time Total difethialone after DFN-Homo-SIDFN-Hetero-SI tube-feeding, DFN-Homo- DFN-Homo- DFN-Hetero- DFN-Hetero-hours dextro laevo dextro laevo 4 4566 5692.5 10589.5 5380.5 9 4692.57141 12155.5 5869.5 24 1243.5 4403.5 8102 3613.5 48 720.5 3874 7974.52804 120 192.5 2087 5431.5 1211 168 129 878.5 3011 392.5 216 77.5 12244030.5 545

In FIG. 9, the relative content of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone in the liver (mean of the contentsmeasured on six rats) expressed in arbitrary units for comparativepurposes is represented by filled circles (●), the relative content ofsaid homo-stereoisomer is represented by empty squares (

) and the relative content of said hetero-stereoisomer is represented byfilled squares (▪). The hepatic content of the dextrorotatory enantiomerof the homo-stereoisomer of difethialone according to the inventiondecreases more rapidly than the hepatic content of saidhomo-stereoisomer of difethialone and than the hepatic content of saidhetero-stereoisomer of difethialone. In addition, the hepatic content ofsaid homo-stereoisomer of difethialone decreases more rapidly than thehepatic content of said hetero-stereoisomer of difethialone. Therepresentation in FIG. 9 takes into account the fact that thedextrorotatory enantiomer of the homo-stereoisomer of difethialoneaccounts for 20% of the difethialone ingested by the rats and that eachof said homo-stereoisomer of difethialone (DFN-Homo-SI) and of saidhetero-stereoisomer of difethialone (DFN-Hetero-SI) accounts for 40% and60%, respectively, of the difethialone ingested by the rats. The hepaticcontent values are related to the percentage of each configurationalstereoisomer in the composition ingested by the rats.

In FIG. 10, the change over time of the content of dextrorotatoryenantiomer of said homo-stereoisomer of difethialone in the liver (meanof the contents measured on six rats expressed in nanograms ofenantiomer per gram of liver (ng/g)) is represented (left-hand scale) byfilled circles (●) and the change over time of the total difethialonecontent is represented (right-hand scale) by empty triangles (Δ). Thehepatic content of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone according to the invention decreasesmore rapidly than the hepatic content of the total difethialone. Therepresentation in FIG. 10 takes into account the fact that thedextrorotatory enantiomer of the homo-stereoisomer of difethialoneaccounts for 20% of the difethialone ingested by the rats and the totaldifethialone accounts for 100% of the difethialone ingested by the rats.

The dextrorotatory enantiomer of the homo-stereoisomer of difethialoneshows significantly lower hepatic persistence relative to all the otherconfigurational stereoisomers of difethialone in the rats.

E. Rodenticidal Bait Comprising a Mass Proportion of 14.7 ppm ofDifethialone

A pasty rodenticidal bait according to the invention is prepared bydispersing an amount of difethialone in an edible excipient comprisingvegetable fat and cereal flour. The measured proportion of difethialonerelative to the bait is 14.7 ppm (14.65 mg of dextrorotatory enantiomerof said homo-stereoisomer of difethialone per kilogram of bait) and theproportion of dextrorotatory enantiomer of said homo-stereoisomerrelative to the difethialone is 99.7%. The bait also comprises a massproportion of 0.3% of laevorotatory enantiomer of saidhetero-stereoisomer of difethialone relative to the difethialone.

On D0, ten coumaphen-sensitive Sprague-Dawley rats (five male and fivefemale SD rats) are placed in individual cages with a rodenticide-freereference feed. On D3, each rat is weighed, and 50 g of rodenticidalbait as described above are then provided to each rat. This provision of50 g of rodenticidal bait is renewed daily. The bait consumed by therats is made up to 50 g of bait on D4, D5 and D6. Starting from D7, theresidual rodenticidal baits are removed and rodenticide-free feed isprovided to all the rats. The rats are monitored for 3 weeks.

The mean amounts of bait consumed daily by a rat at D4, D5, D6 and D7expressed in grams per day are given in table 2 below.

TABLE 2 Bait consumed, g Mean Standard deviation D4 17.8 5.7 D5 16.0 4.7D6 14.2 4.1 D7 9.4 3.8

It should be noted that no rat consumed a daily amount of bait of lessthan 1 g/day. All the rats (100%) die between D9 and D10. The mortalityis 100% on D10. The mean bait consumption, expressed as grams of baitper kilogram of rat, is 50.5 g of bait.

The hepatic content of the dextrorotatory enantiomer of thehomo-stereoisomer of difethialone (“DFN-Homo-dextro”) according to theinvention for each of the rats which die between D9 and D10 is measuredby high-pressure liquid chromatography analysis on a chiral column. Thevalues, expressed as micrograms of “DFN-Homo-dextro” and difethialoneper gram of liver, are given in table 3 below. The mean amount of“DFN-Homo-dextro” enantiomer retained in the liver of each rodent (maleand female) on their death corresponds to 4.7% of the amount ofdifethialone ingested by each rodent.

TABLE 3 Hepatic content, μg/g DFN-Homo-dextro Total difethialone Mean6.1 6.1 Standard deviation 4.5 4.5

For comparative purposes, the difethialone content retained in the liverof rats which consumed a bait comprising 15 ppm of difethialone of theprior art is about 110 μg/gram.

The bait containing a 14.7 ppm dose of difethialone makes it possible toobtain a mortality rate of 100% while minimizing the risks of secondaryintoxication of animals—especially birds—which prey or carrion-feed onweakened target rodent pests that have consumed a rodenticidal bait.

It goes without saying that the invention may be the subject of numerousimplementation variants and applications. In particular, a composition,a rodenticidal bait and a process for controlling target rodent pestsare subject to an infinite number of variants both in the formulation ofthe bait and in the embodiments of the process.

1. Rodenticidal bait comprising at least one excipient that is ediblefor target rodent pests and difethialone in the form of a dextrorotatoryenantiomer of a configurational stereoisomer of difethialone, namedhomo-stereoisomer, the formula of which is3-(4′-bromobiphenyl-4-yl)-1-(4-hydroxythiocoumarin-3-yl)-1,2,3,4-tetrahydronaphthalene,in which carbons 1 and 3 of the 1,2,3,4-tetrahydronaphthalene group ofsaid homo-stereoisomer have the same absolute configuration, with theexclusion of a racemic mixture of the dextrorotatory and laevorotatoryenantiomers of said homo-stereoisomer of difethialone.
 2. Bait accordingto claim 1, wherein said homo-stereoisomer is predominantly indextrorotatory enantiomer form.
 3. Bait according to claim 1, whereinthe difethialone is predominantly in the dextrorotatory enantiomer formof said homo-stereoisomer of difethialone.
 4. Bait according to claim 1,further comprising an amount of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone in the rodenticidal bait such that theratio of this amount to the amount of difethialone in the rodenticidalbait is greater than 25%.
 5. Bait according to claim 1, furthercomprising an amount of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone in the rodenticidal bait such that theratio of this amount to the amount of difethialone in the rodenticidalbait is greater than 97%.
 6. Bait according to claim 1, wherein theedible excipient comprises at least one food chosen from the groupformed from cereal seeds, cereal seed meals, cereal seed flours, cerealseed flakes, cereal bran and non-cereal seeds.
 7. Bait according toclaim 1, further comprising a mass amount of difethialone such that theratio of this mass amount of difethialone to the mass amount ofrodenticidal bait is less than 200 ppm.
 8. Bait according to claim 2,wherein the difethialone is predominantly in the dextrorotatoryenantiomer form of said homo-stereoisomer of difethialone.
 9. Baitaccording to claim 2, further comprising an amount of the dextrorotatoryenantiomer of said homo-stereoisomer of difethialone in the rodenticidalbait such that the ratio of this amount to the amount of difethialone inthe rodenticidal bait is greater than 25%.
 10. Bait according to claim3, further comprising an amount of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone in the rodenticidal bait such that theratio of this amount to the amount of difethialone in the rodenticidalbait is greater than 25%.
 11. Bait according to claim 2, furthercomprising an amount of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone in the rodenticidal bait such that theratio of this amount to the amount of difethialone in the rodenticidalbait is greater than 97%.
 12. Bait according to claim 3, furthercomprising an amount of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone in the rodenticidal bait such that theratio of this amount to the amount of difethialone in the rodenticidalbait is greater than 97%.
 13. Bait according to claim 4, furthercomprising an amount of the dextrorotatory enantiomer of saidhomo-stereoisomer of difethialone in the rodenticidal bait such that theratio of this amount to the amount of difethialone in the rodenticidalbait is greater than 97%.
 14. Bait according to claim 2, wherein theedible excipient comprises at least one food chosen from the groupformed from cereal seeds, cereal seed meals, cereal seed flours, cerealseed flakes, cereal bran and non-cereal seeds.
 15. Bait according toclaim 3, wherein the edible excipient comprises at least one food chosenfrom the group formed from cereal seeds, cereal seed meals, cereal seedflours, cereal seed flakes, cereal bran and non-cereal seeds.
 16. Baitaccording to claim 4, wherein the edible excipient comprises at leastone food chosen from the group formed from cereal seeds, cereal seedmeals, cereal seed flours, cereal seed flakes, cereal bran andnon-cereal seeds.
 17. Bait according to claim 5, wherein the edibleexcipient comprises at least one food chosen from the group formed fromcereal seeds, cereal seed meals, cereal seed flours, cereal seed flakes,cereal bran and non-cereal seeds.
 18. Bait according to claim 2, furthercomprising a mass amount of difethialone such that the ratio of thismass amount of difethialone to the mass amount of rodenticidal bait isless than 200 ppm.
 19. Bait according to claim 3, further comprising amass amount of difethialone such that the ratio of this mass amount ofdifethialone to the mass amount of rodenticidal bait is less than 200ppm.
 20. Bait according to claim 4, further comprising a mass amount ofdifethialone such that the ratio of this mass amount of difethialone tothe mass amount of rodenticidal bait is less than 200 ppm.